A study to examine the amino acids associated with the substrate binding sites in Lactobacillus casei thymidylate synthetase will be undertaken. Various folate analogues will be covalently fixed to the protein in the presence and absence of deoxyuridine 5'-phosphate and 5-fluoro-deoxyuridine 5'-phosphate to determine whether these nucleotides promote the specificity of binding. The peptides containing bound folate will be isolated and sequenced according to methods previously described by us. From this information it should be possible to locate the folate binding site(s) within the linear sequence of thymidylate synthetase and to compare its position within the sequence with what has already been established for 5-fluoro-deoxyuridine 5'-phosphate. Similar studies will be conducted with the folate analogue, methotrexate which will also be fixed to the synthetase and its location established by peptide isolation and sequencing. Treatment of the enzyme with amino acid modifying agents should also provide information on the relationship of specific amino acids to both the binding reaction and their involvement in the mechanistics of the reaction. A comparative study of thymidylate synthetase isolated from Lactobacillus casei, Eschericia coli B, T2 bacteriophage induced E. coli B, Ehrlich ascites cells, HeLa cells, and calf thymus will be conducted. This shall include molecular weight, amino acid analysis, peptide mapping and end group analysis.